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New Compound Anti Mycobacterium tuberculosis from Methanolic Fraction of

Bangle Rhizome (Zingiber cassumunar Roxb.)

Article  in  International Journal of PharmTech Research · January 2017

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 International Journal of PharmTech Research
CODEN (USA): IJPRIF, ISSN: 0974-4304, ISSN(Online): 2455-9563
Vol.10, No.1, pp 57-61, 2017

New Compound Anti Mycobacterium tuberculosis from

Methanolic Fraction of Bangle Rhizome
(Zingiber cassumunar Roxb.)
Muhammad Isrul*,l, Gemini Alam2, Sartini2, Nasrum Massi3
1
Pharmacy Department, STIKES Mandala Waluya, Kendari-Indonesia,
2
Pharmacy Faculty, Hasanuddin University, Makassar-Indonesia,
3
Medical Faculty, Hasanuddin University, Makassar-Indonesia

Abstract : Tuberculosis (TB) remains a major global health problem, responsible for ill health
among millions of people each year.One of the main problem of TB treatment is the resistance
of Mycobacterium tuberculosis. Continuous efforts to develop new drugs to combat M.
Tuberculosisare under way and bioactive compounds of natural origin, particularly from plants,
are gaining significance. Bangle Rhizome (Zingiber cassumuna rRoxb.) has been used
empirically as anti Mycobacterium tuberculosis drug. The aim of researchto determineanti
M.tuberculosis Strain H37Rv and MDR activity of methanol extract fraction of bangle rhizome
(Zingiber cassumunar roxb.). powdered Bangle Rhizome were macerated with hexane and
methanol, respectively. Methanol extract then was fractionated by vacuum liquid
chromatography. Anti Mycobacterium tuberculosis assay was done with Microscopic
ObservationDrugSuspectibility (MODS) method and at final concentration at 100 ppm of
fractions. Methanolextract of bangle rhizome resulting fivefactions (Fraction A,B,C,D and E).
M. tuberculosis assay results show that the most active fraction is B. Chemical screening of
fraction B using TLC visualization reagents indicated that fraction B contains flavonoids and
terpenes.
Keyword : Zingiber cassumunar Roxb., Mycobacterium tuberculosis, MODS.

Introduction
Tuberculosis (TB) remains a major global health problem, responsible for ill health among millions of
people each year. TB ranks as the second leading cause of death from an infectious disease worldwide. In 2013,
there were an estimated 9.0 million incident cases of TB and 1.5 million people died from the disease (1.1
million deaths among people who were HIV-negative and 360 000 among people who were HIV-positive).
Among these deaths there were an estimated 210 000 from MDR-TB, a relatively high total compared with 480
000 incident cases of MDR-TB1. therefore, there is a need to develop new drugs to combat M. tuberculosis,
especially multi-drug resistant (MDR) strain, and continuous efforts are under way in the search for novel
bioactive compounds to develop new anti-tuberculosis drugs. To this end, bioactive compounds of natural
origin, particularly from plants, are gaining significance2.

Bangle (Zingiber Cassumunar Roxb.) is one of the plants used as traditional medicine. Bangle rhizome
have been shown to have several pharmacological effects such as aanti-inflammatory, antibacterial, and
analgetic3,4,5. Previousstudies have been conducted to test the activity ofthe methanol dan hexane extract of
Muhammad Isrul et al /International Journal of PharmTech Research, 2017,10(1): 57-61. 58

bangle rhizomeand The Result show that hexane and methanol extract haveM. Tuberculosis strains H37Rv and
MDR inhibitory activity and methanol extractis the the most active extract6.

The present study was aimed to assay the anti M.tuberculosis Strain H37Rv and MDR activity of
methanol extract fraction of bangle rhizome. (Zingiber cassumunar roxb.)

Materials and Methods

Plant material

Bangle Rhizome (Zingiber cassumunar Roxb.) were obtained from sungguminasa garden, Gowa,
South Sulawesi, Indonesia. The Plant Material was determination by Botany Laboratory, Department of
Biology, Matematics and Science Faculty Hasanuddin University.

Extraction and Fraction

Bangle that have been dried and powdered were macerated with technical grade of hexane and
methanol, respectively. Methanol extract then was fractionated by vacuum liquid chromatography with hexane,
hexane-ethyl acetate, ethyl acetate, and methanol as eluent.

Bacterial strains and inoculum preparation

M. tuberculosis strains H37RvandMDR were supplied by Microbiology Laboratory, medical faculty of

Hasanuddin University. Allcultures were grown in Middle brook 7H9 liquid medium fortified with oleic acid
complex of bovine serum albumin-dextrose-catalase (OADC) at 37°C and agitated once a day for 2 weeks. The
inoculum suspension was made in Phosphate Buffer Solution in turbidity Standard of No.0.5McFarland.

Anti Mycobacterium assay

Anti Mycobacterium Assay was conducted using MODS(Microscopic Observation Drug Suspectibility)
method according Anitaetal7 with modification.The MODS media were prepared in 24-well tissue-culture plates.
Each well contained 950 μl of M.tuberculosis inoculum, Middlebrook 7H9 broth, oxalic acid, albumin, dextrose,
and catalase (OADC), and polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA).
Fifty microliters extract stock solutions were added to give the final extract concentrations to be 100 ppm. The
negative control was DMSO. The rest of the well is used to growth control containing bacteria and media only.
The cultures were examined under an inverted light microscope at a magnification of 10× every day, from day 7
to day 15. To minimize cross-contamination and occupational exposure, plates were permanently sealed inside
plastic zip lock bags after inoculation and were subsequently examined within the bag. The growth of
M.tuberculosis was identified by cord formation (Figure 1).

Figure1. Cord formation of M.tuberculosis

Muhammad Isrul et al /International Journal of PharmTech Research, 2017,10(1): 57-61. 59

Result and Discussion

Fractionation Result of methanol extract of bangle rhizome produced 20 fractions that group into five
factions (Fraction A, B, C,D and E) based on thin layer chromatography(TLC) profile. The fractions were then
tested the activity of antiM. Tuberculosis with MODS method.

Observations of anti M. Tuberculosis assay with MODS method was begun on day 7 to determine the
active fraction. Observations carried out by formations cord of M. tuberculosis. On the 14th day of observation
(Table 1) shows the fraction B as the most active fraction.

Table 1 : Anti mycobacterium assay of methanol extract fraction of Bangle Rhizome.

Sample Replication M.tuberculosis

H37Rv MDR
Fraction A 100 ppm 1 - -
2 + +
3 - +
Fraction B 100 ppm 1 - -
2 - -
3 - -
Fraction C 100 ppm 1 - +
2 - -
3 - -
Fraction D 100 ppm 1 + +
2 + +
3 + +
Fraction E 100 ppm 1 + +
2 + +
3 + +
Negative control 1 + +
(DMSO) 2 + +
3 + +
Control 1 + +
(M.tuberculosis) 2 + +
3 + +
+ = positive of M.tuberculosis growth, - = negative of M.tuberculosis growth

The Result of chemical screening compound of fraction B was done by using TLC visualization
reagents (Table 2) to determine the group of compounds which contained in fraction B. The Results showed
Fraction B positive in FeCl3, Sitoborat, and the Lieberman Burchard reagent showed blue green color spot. The
results indicated that fraction B contains flavonoids and terpenes.

Several studies have reported flavonoids and terpenes compounds as anti M. tuberculosis. One of
Terpenes are reported to be as anti M.tuberculosisis labdan diterpene compound isolated from the Curcuma
amadarhizome8. Sesquiterpene compounds Dihydro-β-agarofuran fromCelastrus vulcancola active against
M.tuberculosis MDR9. The Others, terpene compounds are caniojane, ent-trachylobane-3-one, ent-
trachylobane-17-al, and leubethanol10,11,12.

Table 2 :Chemical screening compound of fraction B

Reagents Result
Dragendorf (-)
FeCl3 (+)
Sitoborat (+)
Liberman Burchard (+)
+ = positive reaction, - = negative reaction
Muhammad Isrul et al /International Journal of PharmTech Research, 2017,10(1): 57-61. 60

Flavonoid Compounds that are reported as anti M.tuberculosis such as Pinocembrin and cryptocaryone,
isolated from the leaf Cryptocarya chinensis Hemsl., which had been proven effective to M.tuberculosis strain
H37Rv13. Other flavonoid compounds are Linaroside and lantanoside isolated from leaf Cryptocarya chinensis,
which as bacteriostatic compound to M.tuberculosis H37Rv14. Sherma et al. Reported that the flavonoid
compound epigallocatechin gallate / epigallocatechin-3-gallate, can inhibit enoyl-acyl carrier protein reductase
an enzyme that plays an important role in the synthesis of fatty acids in bacteria which is generally used as a
cell wall constituent, particularly M. tuberculosis which contain high fatty acid in its cell wall15,16.

Test results showed the content of some of the compounds that allow the compounds to act as anti
M.tuberculosis strains H37Rv and MDR. Therefore, further research is needed to isolate the active compound.

Conclusion
Methanolic Fraction of Bangle Rhizome (Zingiber Cassumunar Roxb.) show anti Mycobacterium
activity against H37Rv and MDR Strain.The Result of chemical screening compound of fractionindicated that
fraction B contains flavonoids and terpenes. Literature investigation shows that flavonoid and terpenes have
been proved as anti Mycobacterium tuberculosis agent. Further research to isolate the compound required to
discover new drugs to combat M. Tuberculosis.

Acknowledgements
The Authors with great full thank to Prof. Elly Wahyuddin, DEA as dean of Faculty of Pharmacy and
Subehan, M.Sc.,Ph.D as the head of master degree program of Pharmacy Faculty of Hasanuddin University,
Indonesia to facilitate for conduct this research.

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